Abstract:

The present invention relates to isolated polypeptides having xylanase
activity and isolated polynucleotides encoding the polypeptides. The
invention also relates to nucleic acid constructs, vectors, and host
cells comprising the polynucleotides as well as methods of producing and
using the polypeptides.

Claims:

1. An isolated polypeptide having xylanase activity, selected from the
group consisting of:(a) a polypeptide comprising an amino acid sequence
having at least 60% identity to the mature polypeptide of SEQ ID NO: 4 or
at least 80% identity to the mature polypeptide of SEQ ID NO: 2;(b) a
polypeptide encoded by a polynucleotide that hybridizes under at least
medium-high stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO. 3, (ii) the genomic DNA sequence comprising the
mature polypeptide coding sequence of SEQ ID NO: 3, or (iii) a
full-length complementary strand of (i) or (ii), or under at least high
stringency conditions with (iv) the mature polypeptide coding sequence of
SEQ ID NO: 1, (v) the genomic DNA sequence comprising the mature
polypeptide coding sequence of SEQ ID NO: 1, or (vi) a full-length
complementary strand of (iv) or (v);(c) a polypeptide encoded by a
polynucleotide comprising a nucleotide sequence having at least 60%
identity to the mature polypeptide coding sequence of SEQ ID NO: 3 or at
least 80% identity to the mature polypeptide coding sequence of SEQ ID
NO: 1; and(d) a variant comprising a substitution, deletion, and/or
insertion of one or more (several) amino acids of the mature polypeptide
of SEQ ID NO: 2 or SEQ ID NO: 4.

2-14. (canceled)

15. The polypeptide of claim 1, comprising or consisting of the amino acid
sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or a fragment thereof having
xylanase activity.

16-33. (canceled)

34. The polypeptide of claim 1, which is encoded by a polynucleotide
comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 or
SEQ ID NO: 3; or a subsequence thereof encoding a fragment having
xylanase activity.

35-37. (canceled)

38. The polypeptide of claim 1, which is encoded by the polynucleotide
contained in plasmid pTter10A which is contained in E. coli NRRL B-50079
or plasmid pTter10B which is contained in E. coli NRRL B-50080.

39-40. (canceled)

41. An isolated polynucleotide comprising a nucleotide sequence that
encodes the polypeptide of claim 1.

42. (canceled)

43. A nucleic acid construct comprising the polynucleotide of claim 41
operably linked to one or more control sequences that direct the
production of the polypeptide in an expression host.

46. A method of producing the polypeptide of claim 1, comprising: (a)
cultivating a cell, which in its wild-type form produces the polypeptide,
under conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide.

47. A method of producing the polypeptide of claim 1, comprising: (a)
cultivating a host cell comprising a nucleic acid construct comprising a
nucleotide sequence encoding the polypeptide under conditions conducive
for production of the polypeptide; and (b) recovering the polypeptide.

48. A method of producing a mutant of a parent cell, comprising disrupting
or deleting a nucleotide sequence encoding the polypeptide of claim 1,
which results in the mutant producing less of the polypeptide than the
parent cell.

49. A mutant cell produced by the method of claim 48.

50-59. (canceled)

60. A method of producing the polypeptide of claim 1, comprising: (a)
cultivating a transgenic plant or a plant cell comprising a
polynucleotide encoding the polypeptide under conditions conducive for
production of the polypeptide; and (b) recovering the polypeptide.

61. A transgenic plant, plant part or plant cell transformed with a
polynucleotide encoding the polypeptide of claim 1.

62. A double-stranded inhibitory RNA (dsRNA) molecule comprising a
subsequence of the polynucleotide of claim 41, wherein optionally the
dsRNA is a siRNA or a miRNA molecule.

63. (canceled)

64. A method of inhibiting the expression of a polypeptide having xylanase
activity in a cell, comprising administering to the cell or expressing in
the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA
comprises a subsequence of the polynucleotide of claim 41.

65. (canceled)

66. A nucleic acid construct comprising a gene encoding a protein operably
linked to a nucleotide sequence encoding a signal peptide comprising or
consisting of amino acids 1 to 19 of SEQ ID NO: 2 or amino acids 1 to 18
of SEQ ID NO: 4, wherein the gene is foreign to the nucleotide sequence.

[0002]This application contains a Sequence Listing in computer readable
form. The computer readable form is incorporated herein by reference.

REFERENCE TO DEPOSITS OF BIOLOGICAL MATERIAL

[0003]This application contains a reference to deposits of biological
material, which deposits are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0005]1. Field of the Invention

[0006]The present invention relates to isolated polypeptides having
xylanase activity and isolated polynucleotides encoding the polypeptides.
The invention also relates to nucleic acid constructs, vectors, and host
cells comprising the polynucleotides as well as methods of producing and
using the polypeptides.

[0007]2. Description of the Related Art

[0008]Plant cell wall polysaccharides constitute approximately 90% of the
plant cell wall and can be divided into three groups: cellulose,
hemicellulose, and pectin. Cellulose represents the major constituent of
call wall polysaccharides. Hemicelluloses are the second most abundant
constituent of plant cell walls. The major hemicellulose polymer is
xylan. The structure of xylans found in cell walls of plants can differ
significantly depending on their origin, but they always contain a
beta-1,4-linked D-xylose backbone. The beta-1,4-linked D-xylose backbone
can be substituted by various side groups, such as L-aribinose,
D-galactose, acetyl, feruloyl, p-coumaroyl, and glucuronic acid residues.

[0009]The biodegradation of the xylan backbone depends on two classes of
enzymes: endoxylanases and beta-xylosidases. Endoxylanases (EC 3.2.1.8)
cleave the xylan backbone into smaller oligosaccharides, which can be
further degraded to xylose by beta-xylosidases (EC 3.2.1.37). Other
enzymes involved in the degradation of xylan include, for example,
acetylxylan esterase, arabinase, alpha-glucuronidase, ferulic acid
esterase, and p-coumaric acid esterase.

[0012]The present invention relates to isolated polypeptides having
xylanase activity selected from the group consisting of:

[0013](a) a polypeptide comprising an amino acid sequence having at least
60% identity to the mature polypeptide of SEQ ID NO: 4 or at least 80%
identity to the mature polypeptide of SEQ ID NO: 2;

[0014](b) a polypeptide encoded by a polynucleotide that hybridizes under
at least medium-high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 3, (ii) the genomic DNA
sequence comprising the mature polypeptide coding sequence of SEQ ID NO:
3, or (iii) a full-length complementary strand of (i) or (ii), or under
at least high stringency conditions with (iv) the mature polypeptide
coding sequence of SEQ ID NO: 1, (v) the genomic DNA sequence comprising
the mature polypeptide coding sequence of SEQ ID NO: 1, or (vi) a
full-length complementary strand of (iv) or (v);

[0015](c) a polypeptide encoded by a polynucleotide comprising a
nucleotide sequence having at least 60% identity to the mature
polypeptide coding sequence of SEQ ID NO: 3 or at least 80% identity to
the mature polypeptide coding sequence of SEQ ID NO: 1; and

[0016](d) a variant comprising a substitution, deletion, and/or insertion
of one or more (several) amino acids of the mature polypeptide of SEQ ID
NO: 2 or SEQ ID NO: 4.

[0017]The present invention also relates to isolated polynucleotides
encoding polypeptides having xylanase activity, selected from the group
consisting of:

[0018](a) a polynucleotide encoding a polypeptide comprising an amino acid
sequence having at least 60% identity to the mature polypeptide of SEQ ID
NO: 4 or at least 80% identity to the mature polypeptide of SEQ ID NO: 2;

[0020](c) a polynucleotide comprising a nucleotide sequence having at
least 60% identity to the mature polypeptide coding sequence of SEQ ID
NO: 3 or at least 80% identity to the mature polypeptide coding sequence
of SEQ ID NO: 1; and

[0021](d) a polynucleotide encoding a variant comprising a substitution,
deletion, and/or insertion of one or more (several) amino acids of the
mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

[0023]The present invention also relates to methods of inhibiting the
expression of a polypeptide having xylanase activity in a cell,
comprising administering to the cell or expressing in the cell a
double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a
subsequence of a polynucleotide of the present invention. The present
also relates to a double-stranded inhibitory RNA (dsRNA) molecule,
wherein optionally the dsRNA is a siRNA or a miRNA molecule.

[0024]The present invention also relates to methods for degrading a
xylan-containing material with a polypeptide having xylanase activity.

[0026]The present invention also relates to methods of producing a
polypeptide having xylanase, comprising: (a) cultivating a transgenic
plant or a plant cell comprising a polynucleotide encoding the
polypeptide having xylanase activity under conditions conducive for
production of the polypeptide; and (b) recovering the polypeptide.

[0027]The present invention further relates to nucleic acid constructs
comprising a gene encoding a protein, wherein the gene is operably linked
to a nucleotide sequence encoding a signal peptide comprising or
consisting of amino acids 1 to 19 of SEQ ID NO: 2 or amino acids 1 to 18
of SEQ ID NO: 4, wherein the gene is foreign to the nucleotide sequence.

[0032]Xylanase activity: The term "xylanase activity" is defined herein as
a 1,4-beta-D-xylan-xylanohydrolase activity (E.C. 3.2.1.8) that catalyzes
the endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For
purposes of the present invention, xylanase activity is determined using
0.2% AZCL-arabinoxylan as substrate in 0.01% Triton X-100 and 200 mM
sodium phosphate pH 6 at 37° C. One unit of xylanase activity is
defined as 1.0 μmole of azurine produced per minute at 37° C.,
pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate
pH 6 buffer.

[0033]The polypeptides of the present invention have at least 20%,
preferably at least 40%, more preferably at least 50%, more preferably at
least 60%, more preferably at least 70%, more preferably at least 80%,
even more preferably at least 90%, most preferably at least 95%, and even
most preferably at least 100% of the xylanase activity of the mature
polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

[0034]Family 10 or Family GH10 or GH10: The term "Family 10" or "Family
GH10" or "GH10" is defined herein as a polypeptide falling into the
glycoside hydrolase Family 10 according to Henrissat B., 1991, A
classification of glycosyl hydrolases based on amino-acid sequence
similarities, Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996,
Updating the sequence-based classification of glycosyl hydrolases,
Biochem. J. 316: 695-696.

[0035]Xylan-containing material: The term "xylan-containing material" is
defined herein as any material comprising xylan as a constituent. Xylan
is a plant cell wall polysaccharide containing a backbone of
beta-1,4-linked xylose residues. Side chains of 4-O-methylglucuronic acid
and arabinose are generally present in varying amounts, together with
acetyl and feruloyl groups. Xylan is a major constituent of
hemicellulose.

[0036]Isolated polypeptide: The term "isolated polypeptide" as used herein
refers to a polypeptide that is isolated from a source. In a preferred
aspect, the polypeptide is at least 1% pure, preferably at least 5% pure,
more preferably at least 10% pure, more preferably at least 20% pure,
more preferably at least 40% pure, more preferably at least 60% pure,
even more preferably at least 80% pure, and most preferably at least 90%
pure, as determined by SDS-PAGE.

[0037]Substantially pure polypeptide: The term "substantially pure
polypeptide" denotes herein a polypeptide preparation that contains at
most 10%, preferably at most 8%, more preferably at most 6%, more
preferably at most 5%, more preferably at most 4%, more preferably at
most 3%, even more preferably at most 2%, most preferably at most 1%, and
even most preferably at most 0.5% by weight of other polypeptide material
with which it is natively or recombinantly associated. It is, therefore,
preferred that the substantially pure polypeptide is at least 92% pure,
preferably at least 94% pure, more preferably at least 95% pure, more
preferably at least 96% pure, more preferably at least 97% pure, more
preferably at least 98% pure, even more preferably at least 99% pure,
most preferably at least 99.5% pure, and even most preferably 100% pure
by weight of the total polypeptide material present in the preparation.
The polypeptides of the present invention are preferably in a
substantially pure form, i.e., that the polypeptide preparation is
essentially free of other polypeptide material with which it is natively
or recombinantly associated. This can be accomplished, for example, by
preparing the polypeptide by well-known recombinant methods or by
classical purification methods.

[0038]Mature polypeptide: The term "mature polypeptide" is defined herein
as a polypeptide in its final form following translation and any
post-translational modifications, such as N-terminal processing,
C-terminal truncation, glycosylation, phosphorylation, etc. In a
preferred aspect, the mature polypeptide is amino acids 20 to 369 of SEQ
ID NO: 2 based on the SignalP software program (Nielsen et al., 1997,
Protein Engineering 10:1-6) that predicts amino acids 1 to 19 of SEQ ID
NO: 2 are a signal peptide. In another preferred aspect, the mature
polypeptide is amino acids 19 to 414 of SEQ ID NO: 4 based on the SignalP
software program that predicts amino acids 1 to 18 of SEQ ID NO: 4 are a
signal peptide.

[0039]Mature polypeptide coding sequence: The term "mature polypeptide
coding sequence" is defined herein as a nucleotide sequence that encodes
a mature polypeptide having xylanase activity. In a preferred aspect, the
mature polypeptide coding sequence is nucleotides 58 to 1107 of SEQ ID
NO: 1 based on the SignalP software program that predicts nucleotides 1
to 57 encode a signal peptide. In another preferred aspect, the mature
polypeptide coding sequence is nucleotides 55 to 1242 of SEQ ID NO: 3
based on the SignalP software program that predicts nucleotides 1 to 54
encode a signal peptide.

[0040]Identity: The relatedness between two amino acid sequences or
between two nucleotide sequences is described by the parameter
"identity".

[0041]For purposes of the present invention, the degree of identity
between two amino acid sequences is determined using the Needleman-Wunsch
algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as
implemented in the Needle program of the EMBOSS package (EMBOSS: The
European Molecular Biology Open Software Suite, Rice et al., 2000, Trends
in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional
parameters used are gap open penalty of 10, gap extension penalty of 0.5,
and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The
output of Needle labeled "longest identity" (obtained using the--nobrief
option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment-Total Number of Gaps
in Alignment)

[0042]For purposes of the present invention, the degree of identity
between two deoxyribonucleotide sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as
implemented in the Needle program of the EMBOSS package (EMBOSS: The
European Molecular Biology Open Software Suite, Rice et al., 2000,
supra), preferably version 3.0.0 or later. The optional parameters used
are gap open penalty of 10, gap extension penalty of 0.5, and the
EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output
of Needle labeled "longest identity" (obtained using the--nobrief option)
is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment-Total
Number of Gaps in Alignment)

[0043]Homologous sequence: The term "homologous sequence" is defined
herein as a predicted protein that has an E value (or expectancy score)
of less than 0.001 in a tfasty search (Pearson, W. R., 1999, in
Bioinformatics Methods and Protocols, S. Misener and S. A. Krawetz, ed.,
pp. 185-219) with the Thielavia terrestris xylanase of SEQ ID NO: 2 or
SEQ ID NO: 4; or the mature polypeptides thereof.

[0044]Polypeptide fragment: The term "polypeptide fragment" is defined
herein as a polypeptide having one or more (several) amino acids deleted
from the amino and/or carboxyl terminus of the mature polypeptide of SEQ
ID NO: 2 or SEQ ID NO: 4; or a homologous sequence thereof; wherein the
fragment has xylanase activity. In a preferred aspect, a fragment
contains at least 305 amino acid residues, more preferably at least 320
amino acid residues, and most preferably at least 335 amino acid
residues, of the mature polypeptide of SEQ ID NO: 2 or a homologous
sequence thereof. In another preferred aspect, a fragment contains at
least 340 amino acid residues, more preferably at least 360 amino acid
residues, and most preferably at least 380 amino acid residues, of the
mature polypeptide of SEQ ID NO: 4 or a homologous sequence thereof.

[0045]Subsequence: The term "subsequence" is defined herein as a
nucleotide sequence having one or more (several) nucleotides deleted from
the 5' and/or 3' end of the mature polypeptide coding sequence of SEQ ID
NO: 1 or SEQ ID NO: 3; or a homologous sequence thereof; wherein the
subsequence encodes a polypeptide fragment having xylanase activity. In a
preferred aspect, a subsequence contains at least 915 nucleotides, more
preferably at least 960 nucleotides, and most preferably at least 1005
nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 1 or
a homologous sequence thereof. In another preferred aspect, a subsequence
contains at least 1020 nucleotides, more preferably at least 1080
nucleotides, and most preferably at least 1140 nucleotides of the mature
polypeptide coding sequence of SEQ ID NO: 3 or a homologous sequence
thereof.

[0046]Allelic variant: The term "allelic variant" denotes herein any of
two or more alternative forms of a gene occupying the same chromosomal
locus. Allelic variation arises naturally through mutation, and may
result in polymorphism within populations. Gene mutations can be silent
(no change in the encoded polypeptide) or may encode polypeptides having
altered amino acid sequences. An allelic variant of a polypeptide is a
polypeptide encoded by an allelic variant of a gene.

[0047]Isolated polynucleotide: The term "isolated polynucleotide" as used
herein refers to a polynucleotide that is isolated from a source. In a
preferred aspect, the polynucleotide is at least 1% pure, preferably at
least 5% pure, more preferably at least 10% pure, more preferably at
least 20% pure, more preferably at least 40% pure, more preferably at
least 60% pure, even more preferably at least 80% pure, and most
preferably at least 90% pure, as determined by agarose electrophoresis.

[0048]Substantially pure polynucleotide: The term "substantially pure
polynucleotide" as used herein refers to a polynucleotide preparation
free of other extraneous or unwanted nucleotides and in a form suitable
for use within genetically engineered protein production systems. Thus, a
substantially pure polynucleotide contains at most 10%, preferably at
most 8%, more preferably at most 6%, more preferably at most 5%, more
preferably at most 4%, more preferably at most 3%, even more preferably
at most 2%, most preferably at most 1%, and even most preferably at most
0.5% by weight of other polynucleotide material with which it is natively
or recombinantly associated. A substantially pure polynucleotide may,
however, include naturally occurring 5' and 3' untranslated regions, such
as promoters and terminators. It is preferred that the substantially pure
polynucleotide is at least 90% pure, preferably at least 92% pure, more
preferably at least 94% pure, more preferably at least 95% pure, more
preferably at least 96% pure, more preferably at least 97% pure, even
more preferably at least 98% pure, most preferably at least 99% pure, and
even most preferably at least 99.5% pure by weight. The polynucleotides
of the present invention are preferably in a substantially pure form,
i.e., that the polynucleotide preparation is essentially free of other
polynucleotide material with which it is natively or recombinantly
associated. The polynucleotides may be of genomic, cDNA, RNA,
semisynthetic, synthetic origin, or any combinations thereof.

[0049]Coding sequence: When used herein the term "coding sequence" means a
nucleotide sequence, which directly specifies the amino acid sequence of
its protein product. The boundaries of the coding sequence are generally
determined by an open reading frame, which usually begins with the ATG
start codon or alternative start codons such as GTG and TTG and ends with
a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA,
cDNA, synthetic, or recombinant nucleotide sequence.

[0050]cDNA: The term "cDNA" is defined herein as a DNA molecule that can
be prepared by reverse transcription from a mature, spliced, mRNA
molecule obtained from a eukaryotic cell. cDNA lacks intron sequences
that may be present in the corresponding genomic DNA. The initial,
primary RNA transcript is a precursor to mRNA that is processed through a
series of steps before appearing as mature spliced mRNA. These steps
include the removal of intron sequences by a process called splicing.
cDNA derived from mRNA lacks, therefore, any intron sequences.

[0051]Nucleic acid construct: The term "nucleic acid construct" as used
herein refers to a nucleic acid molecule, either single- or
double-stranded, which is isolated from a naturally occurring gene or
which is modified to contain segments of nucleic acids in a manner that
would not otherwise exist in nature or which is synthetic. The term
nucleic acid construct is synonymous with the term "expression cassette"
when the nucleic acid construct contains the control sequences required
for expression of a coding sequence of the present invention.

[0052]Control sequences: The term "control sequences" is defined herein to
include all components, which are necessary or advantageous for the
expression of a polynucleotide encoding a polypeptide of the present
invention. Each control sequence may be native or foreign to the
nucleotide sequence encoding the polypeptide or native or foreign to each
other. Such control sequences include, but are not limited to, a leader,
polyadenylation sequence, propeptide sequence, promoter, signal peptide
sequence, and transcription terminator. At a minimum, the control
sequences include a promoter, and transcriptional and translational stop
signals. The control sequences may be provided with linkers for the
purpose of introducing specific restriction sites facilitating ligation
of the control sequences with the coding region of the nucleotide
sequence encoding a polypeptide.

[0053]Operably linked: The term "operably linked" denotes herein a
configuration in which a control sequence is placed at an appropriate
position relative to the coding sequence of the polynucleotide sequence
such that the control sequence directs the expression of the coding
sequence of a polypeptide.

[0054]Expression: The term "expression" includes any step involved in the
production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.

[0055]Expression vector: The term "expression vector" is defined herein as
a linear or circular DNA molecule that comprises a polynucleotide
encoding a polypeptide of the present invention and is operably linked to
additional nucleotides that provide for its expression.

[0056]Host cell: The term "host cell", as used herein, includes any cell
type that is susceptible to transformation, transfection, transduction,
and the like with a nucleic acid construct or expression vector
comprising a polynucleotide of the present invention.

[0057]Modification: The term "modification" means herein any chemical
modification of the polypeptide consisting of the mature polypeptide of
SEQ ID NO: 2 or SEQ ID NO: 4; or a homologous sequence thereof; as well
as genetic manipulation of the DNA encoding such a polypeptide. The
modification can be a substitution, a deletion, and/or an insertion of
one or more (several) amino acids as well as replacements of one or more
(several) amino acid side chains.

[0058]Artificial variant: When used herein, the term "artificial variant"
means a polypeptide having xylanase activity produced by an organism
expressing a modified polynucleotide sequence of the mature polypeptide
coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or a homologous sequence
thereof. The modified nucleotide sequence is obtained through human
intervention by modification of the polynucleotide sequence disclosed in
SEQ ID NO: 1 or SEQ ID NO: 3; or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Xylanase Activity

[0059]In a first aspect, the present invention relates to isolated
polypeptides comprising an amino acid sequence having a degree of
identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 of
preferably at least 60%, more preferably at least 65%, more preferably at
least 70%, more preferably at least 75%, more preferably at least 80%,
more preferably at least 85%, even more preferably at least 90%, most
preferably at least 95%, and even most preferably at least 96%, at least
97%, at least 98%, or at least 99%, which have xylanase activity
(hereinafter "homologous polypeptides"). In a preferred aspect, the
homologous polypeptides have an amino acid sequence that differs by ten
amino acids, preferably by five amino acids, more preferably by four
amino acids, even more preferably by three amino acids, most preferably
by two amino acids, and even most preferably by one amino acid from the
mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

[0061]In a preferred aspect, the polypeptide comprises the amino acid
sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide
comprises the mature polypeptide of SEQ ID NO: 2. In another preferred
aspect, the polypeptide comprises amino acids 20 to 369 of SEQ ID NO: 2,
or an allelic variant thereof; or a fragment thereof having xylanase
activity. In another preferred aspect, the polypeptide comprises amino
acids 20 to 369 of SEQ ID NO: 2. In another preferred aspect, the
polypeptide consists of the amino acid sequence of SEQ ID NO: 2 or an
allelic variant thereof; or a fragment thereof having xylanase activity.
In another preferred aspect, the polypeptide consists of the amino acid
sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide
consists of the mature polypeptide of SEQ ID NO: 2. In another preferred
aspect, the polypeptide consists of amino acids 20 to 369 of SEQ ID NO: 2
or an allelic variant thereof; or a fragment thereof having xylanase
activity. In another preferred aspect, the polypeptide consists of amino
acids 20 to 369 of SEQ ID NO: 2.

[0062]In another preferred aspect, the polypeptide comprises the amino
acid sequence of SEQ ID NO: 4. In another preferred aspect, the
polypeptide comprises the mature polypeptide of SEQ ID NO: 4. In another
preferred aspect, the polypeptide comprises amino acids 19 to 414 of SEQ
ID NO: 4, or an allelic variant thereof; or a fragment thereof having
xylanase activity. In another preferred aspect, the polypeptide comprises
amino acids 19 to 414 of SEQ ID NO: 4. In another preferred aspect, the
polypeptide consists of the amino acid sequence of SEQ ID NO: 4 or an
allelic variant thereof; or a fragment thereof having xylanase activity.
In another preferred aspect, the polypeptide consists of the amino acid
sequence of SEQ ID NO: 4. In another preferred aspect, the polypeptide
consists of the mature polypeptide of SEQ ID NO: 4. In another preferred
aspect, the polypeptide consists of amino acids 19 to 414 of SEQ ID NO: 4
or an allelic variant thereof; or a fragment thereof having xylanase
activity. In another preferred aspect, the polypeptide consists of amino
acids 19 to 414 of SEQ ID NO: 4.

[0064]The nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or a
subsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2
or SEQ ID NO: 4; or a fragment thereof; may be used to design nucleic
acid probes to identify and clone DNA encoding polypeptides having
xylanase activity from strains of different genera or species according
to methods well known in the art. In particular, such probes can be used
for hybridization with the genomic or cDNA of the genus or species of
interest, following standard Southern blotting procedures, in order to
identify and isolate the corresponding gene therein. Such probes can be
considerably shorter than the entire sequence, but should be at least 14,
preferably at least 25, more preferably at least 35, and most preferably
at least 70 nucleotides in length. It is, however, preferred that the
nucleic acid probe is at least 100 nucleotides in length. For example,
the nucleic acid probe may be at least 200 nucleotides, preferably at
least 300 nucleotides, more preferably at least 400 nucleotides, or most
preferably at least 500 nucleotides in length. Even longer probes may be
used, e.g., nucleic acid probes that are preferably at least 600
nucleotides, more preferably at least 700 nucleotides, even more
preferably at least 800 nucleotides, or most preferably at least 900
nucleotides in length. Both DNA and RNA probes can be used. The probes
are typically labeled for detecting the corresponding gene (for example,
with 32P, 3H, 35S, biotin, or avidin). Such probes are
encompassed by the present invention.

[0065]A genomic DNA or cDNA library prepared from such other strains may,
therefore, be screened for DNA that hybridizes with the probes described
above and encodes a polypeptide having xylanase activity. Genomic or
other DNA from such other strains may be separated by agarose or
polyacrylamide gel electrophoresis, or other separation techniques. DNA
from the libraries or the separated DNA may be transferred to and
immobilized on nitrocellulose or other suitable carrier material. In
order to identify a clone or DNA that is homologous with SEQ ID NO: 1 or
SEQ ID NO: 3; or a subsequence thereof; the carrier material is
preferably used in a Southern blot.

[0066]For purposes of the present invention, hybridization indicates that
the nucleotide sequence hybridizes to a labeled nucleic acid probe
corresponding to the mature polypeptide coding sequence of SEQ ID NO: 1
or SEQ ID NO: 3; the genomic DNA sequence comprising the mature
polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3; its
full-length complementary strand; or a subsequence thereof; under very
low to very high stringency conditions. Molecules to which the nucleic
acid probe hybridizes under these conditions can be detected using, for
example, X-ray film.

[0067]In a preferred aspect, the nucleic acid probe is the mature
polypeptide coding sequence of SEQ ID NO: 1. In another preferred aspect,
the nucleic acid probe is nucleotides 58 to 1107 of SEQ ID NO: 1. In
another preferred aspect, the nucleic acid probe is a polynucleotide
sequence that encodes the polypeptide of SEQ ID NO: 2, or a subsequence
thereof. In another preferred aspect, the nucleic acid probe is SEQ ID
NO: 1. In another preferred aspect, the nucleic acid probe is the
polynucleotide sequence contained in plasmid pTter10A which is contained
in E. coli NRRL B-50079, wherein the polynucleotide sequence thereof
encodes a polypeptide having xylanase activity. In another preferred
aspect, the nucleic acid probe is the mature polypeptide coding region
contained in plasmid pTter10A which is contained in E. coli NRRL B-50079.

[0068]In another preferred aspect, the nucleic acid probe is the mature
polypeptide coding sequence of SEQ ID NO: 3. In another preferred aspect,
the nucleic acid probe is nucleotides 55 to 1242 of SEQ ID NO: 3. In
another preferred aspect, the nucleic acid probe is a polynucleotide
sequence that encodes the polypeptide of SEQ ID NO: 4, or a subsequence
thereof. In another preferred aspect, the nucleic acid probe is SEQ ID
NO: 3. In another preferred aspect, the nucleic acid probe is the
polynucleotide sequence contained in plasmid pTter10B which is contained
in E. coli NRRL B-50080, wherein the polynucleotide sequence thereof
encodes a polypeptide having xylanase activity. In another preferred
aspect, the nucleic acid probe is the mature polypeptide coding region
contained in plasmid pTter10B which is contained in E. coli NRRL B-50080.

[0069]For long probes of at least 100 nucleotides in length, very low to
very high stringency conditions are defined as prehybridization and
hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml
sheared and denatured salmon sperm DNA, and either 25% formamide for very
low and low stringencies, 35% formamide for medium and medium-high
stringencies, or 50% formamide for high and very high stringencies,
following standard Southern blotting procedures for 12 to 24 hours
optimally.

[0070]For long probes of at least 100 nucleotides in length, the carrier
material is finally washed three times each for 15 minutes using
2×SSC, 0.2% SDS preferably at 45° C. (very low stringency),
more preferably at 50° C. (low stringency), more preferably at
55° C. (medium stringency), more preferably at 60° C.
(medium-high stringency), even more preferably at 65° C. (high
stringency), and most preferably at 70° C. (very high stringency).

[0072]For short probes of about 15 nucleotides to about 70 nucleotides in
length, the carrier material is washed once in 6×SCC plus 0.1% SDS
for 15 minutes and twice each for 15 minutes using 6×SSC at
5° C. to 10° C. below the calculated Tm.

[0073]In a third aspect, the present invention relates to isolated
polypeptides having xylanase activity encoded by polynucleotides
comprising or consisting of nucleotide sequences that have a degree of
identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ
ID NO: 3 of preferably at least 60%, more preferably at least 65%, more
preferably at least 70%, more preferably at least 75%, more preferably at
least 80%, more preferably at least 85%, even more preferably at least
90%, most preferably at least 95%, and even most preferably at least 96%,
at least 97%, at least 98%, or at least 99%, which encode a polypeptide
having xylanase activity. See polynucleotide section herein.

[0074]In a fourth aspect, the present invention relates to artificial
variants comprising a substitution, deletion, and/or insertion of one or
more (or several) amino acids of the mature polypeptide of SEQ ID NO: 2
or SEQ ID NO: 4; or a homologous sequence thereof. Preferably, amino acid
changes are of a minor nature, that is conservative amino acid
substitutions or insertions that do not significantly affect the folding
and/or activity of the protein; small deletions, typically of one to
about 30 amino acids; small amino- or carboxyl-terminal extensions, such
as an amino-terminal methionine residue; a small linker peptide of up to
about 20-25 residues; or a small extension that facilitates purification
by changing net charge or another function, such as a poly-histidine
tract, an antigenic epitope or a binding domain.

[0076]In addition to the 20 standard amino acids, non-standard amino acids
(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,
isovaline, and alpha-methyl serine) may be substituted for amino acid
residues of a wild-type polypeptide. A limited number of non-conservative
amino acids, amino acids that are not encoded by the genetic code, and
unnatural amino acids may be substituted for amino acid residues.
"Unnatural amino acids" have been modified after protein synthesis,
and/or have a chemical structure in their side chain(s) different from
that of the standard amino acids. Unnatural amino acids can be chemically
synthesized, and preferably, are commercially available, and include
pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and
4-methylproline, and 3,3-dimethylproline.

[0077]Alternatively, the amino acid changes are of such a nature that the
physico-chemical properties of the polypeptides are altered. For example,
amino acid changes may improve the thermal stability of the polypeptide,
alter the substrate specificity, change the pH optimum, and the like.

[0078]Essential amino acids in the parent polypeptide can be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,
Science 244: 1081-1085). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological activity (i.e.,
xylanase activity) to identify amino acid residues that are critical to
the activity of the molecule. See also, Hilton et al., 1996, J. Biol.
Chem. 271: 4699-4708. The active site of the enzyme or other biological
interaction can also be determined by physical analysis of structure, as
determined by such techniques as nuclear magnetic resonance,
crystallography, electron diffraction, or photoaffinity labeling, in
conjunction with mutation of putative contact site amino acids. See, for
example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,
J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 5964.
The identities of essential amino acids can also be inferred from
analysis of identities with polypeptides that are related to a
polypeptide according to the invention.

[0080]Mutagenesis/shuffling methods can be combined with high-throughput,
automated screening methods to detect activity of cloned, mutagenized
polypeptides expressed by host cells (Ness et al., 1999, Nature
Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active
polypeptides can be recovered from the host cells and rapidly sequenced
using standard methods in the art. These methods allow the rapid
determination of the importance of individual amino acid residues in a
polypeptide of interest, and can be applied to polypeptides of unknown
structure.

[0081]The total number of amino acid substitutions, deletions and/or
insertions of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4,
such as amino acids 20 to 369 of SEQ ID NO: 2 or amino acids 17 to 413 of
SEQ ID NO:4, is 10, preferably 9, more preferably 8, more preferably 7,
more preferably at most 6, more preferably 5, more preferably 4, even
more preferably 3, most preferably 2, and even most preferably 1.

Sources of Polypeptides Having Xylanase Activity

[0082]A polypeptide of the present invention may be obtained from
microorganisms of any genus. For purposes of the present invention, the
term "obtained from" as used herein in connection with a given source
shall mean that the polypeptide encoded by a nucleotide sequence is
produced by the source or by a strain in which the nucleotide sequence
from the source has been inserted. In a preferred aspect, the polypeptide
obtained from a given source is secreted extracellularly.

[0083]A polypeptide having xylanase activity of the present invention may
be a bacterial polypeptide. For example, the polypeptide may be a gram
positive bacterial polypeptide such as a Bacillus, Streptococcus,
Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus,
Clostridium, Geobacillus, or Oceanobacillus polypeptide having xylanase
activity, or a Gram negative bacterial polypeptide such as an E. coli,
Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,
Fusobacterium, llyobacter, Neisseria, or Ureaplasma polypeptide having
xylanase activity.

[0091]In a more preferred aspect, the polypeptide is a Thielavia
terrestris polypeptide having xylanase activity. In a most preferred
aspect, the polypeptide is a Thielavia terrestris NRRL 8126 polypeptide
having xylanase activity, e.g., the polypeptide comprising the mature
polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

[0092]It will be understood that for the aforementioned species the
invention encompasses both the perfect and imperfect states, and other
taxonomic equivalents, e.g., anamorphs, regardless of the species name by
which they are known. Those skilled in the art will readily recognize the
identity of appropriate equivalents.

[0093]Strains of these species are readily accessible to the public in a
number of culture collections, such as the American Type Culture
Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural
Research Service Patent Culture Collection, Northern Regional Research
Center (NRRL).

[0094]Furthermore, such polypeptides may be identified and obtained from
other sources including microorganisms isolated from nature (e.g., soil,
composts, water, etc.) using the above-mentioned probes. Techniques for
isolating microorganisms from natural habitats are well known in the art.
The polynucleotide may then be obtained by similarly screening a genomic
or cDNA library of such a microorganism. Once a polynucleotide sequence
encoding a polypeptide has been detected with the probe(s), the
polynucleotide can be isolated or cloned by utilizing techniques that are
well known to those of ordinary skill in the art (see, e.g., Sambrook et
al., 1989, supra).

[0095]Polypeptides of the present invention also include fused
polypeptides or cleavable fusion polypeptides in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide or fragment thereof. A fused polypeptide is produced by
fusing a nucleotide sequence (or a portion thereof) encoding another
polypeptide to a nucleotide sequence (or a portion thereof of the present
invention. Techniques for producing fusion polypeptides are known in the
art, and include ligating the coding sequences encoding the polypeptides
so that they are in frame and that expression of the fused polypeptide is
under control of the same promoter(s) and terminator.

[0096]A fusion polypeptide can further comprise a cleavage site. Upon
secretion of the fusion protein, the site is cleaved releasing the
polypeptide having xylanase activity from the fusion protein. Examples of
cleavage sites include, but are not limited to, a Kex2 site that encodes
the dipeptide Lys-Arg (Martin et al., 2003, J. Ind. Microbiol.
Biotechnol. 3: 568-76; Svetina et al., 2000, J. Biotechnol. 76: 245-251;
Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493;
Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991,
Biotechnology 9: 378-381), an Ile-(Glu or Asp)-Gly-Arg site, which is
cleaved by a Factor Xa protease after the arginine residue (Eaton et al.,
1986, Biochem. 25: 505-512); a Asp-Asp-Asp-Asp-Lys site, which is cleaved
by an enterokinase after the lysine (Collins-Racie et al., 1995,
Biotechnology 13: 982-987); a His-Tyr-Glu site or His-Tyr-Asp site, which
is cleaved by Genenase I (Carter et al., 1989, Proteins: Structure,
Function, and Genetics 6: 240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which
is cleaved by thrombin after the Arg (Stevens, 2003, Drug Discovery World
4: 35-48); a Glu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV
protease after the Gln (Stevens, 2003, supra); and a
Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro site, which is cleaved by a genetically
engineered form of human rhinovirus 3C protease after the Gln (Stevens,
2003, supra).

Polynucleotides

[0097]The present invention also relates to isolated polynucleotides
comprising or consisting of nucleotide sequences that encode polypeptides
having xylanase activity of the present invention.

[0098]In a preferred aspect, the nucleotide sequence comprises or consists
of SEQ ID NO: 1. In another more preferred aspect, the nucleotide
sequence comprises or consists of the sequence contained in plasmid
pTter10A which is contained in E. coli NRRL B-50079. In another preferred
aspect, the nucleotide sequence comprises or consists of the mature
polypeptide coding sequence of SEQ ID NO: 1. In another preferred aspect,
the nucleotide sequence comprises or consists of nucleotides 58 to 1107
of SEQ ID NO: 1. In another more preferred aspect, the nucleotide
sequence comprises or consists of the mature polypeptide coding sequence
contained in plasmid pTter10A which is contained in E. coli NRRL B-50079.
The present invention also encompasses nucleotide sequences that encode
polypeptides comprising or consisting of the amino acid sequence of SEQ
ID NO: 2 or the mature polypeptide thereof, which differ from SEQ ID NO:
1 or the mature polypeptide coding sequence thereof by virtue of the
degeneracy of the genetic code. The present invention also relates to
subsequences of SEQ ID NO: 1 that encode fragments of SEQ ID NO: 2 that
have xylanase activity.

[0099]In another preferred aspect, the nucleotide sequence comprises or
consists of SEQ ID NO: 3. In another more preferred aspect, the
nucleotide sequence comprises or consists of the sequence contained in
plasmid pTter10B which is contained in E. coli NRRL B-50080. In another
preferred aspect, the nucleotide sequence comprises or consists of the
mature polypeptide coding sequence of SEQ ID NO: 3. In another preferred
aspect, the nucleotide sequence comprises or consists of nucleotides 55
to 1242 of SEQ ID NO: 3. In another more preferred aspect, the nucleotide
sequence comprises or consists of the mature polypeptide coding sequence
contained in plasmid pTter10B which is contained in E. coli NRRL B-50080.
The present invention also encompasses nucleotide sequences that encode
polypeptides comprising or consisting of the amino acid sequence of SEQ
ID NO: 4 or the mature polypeptide thereof, which differ from SEQ ID NO:
3 or the mature polypeptide coding sequence thereof by virtue of the
degeneracy of the genetic code. The present invention also relates to
subsequences of SEQ ID NO: 3 that encode fragments of SEQ ID NO: 4 that
have xylanase activity.

[0100]The present invention also relates to mutant polynucleotides
comprising or consisting of at least one mutation in the mature
polypeptide coding sequence of SEQ ID NO: 1 and SEQ ID NO: 3, in which
the mutant nucleotide sequence encodes the mature polypeptide of SEQ ID
NO: 2 and SEQ ID NO: 4, respectively.

[0101]The techniques used to isolate or clone a polynucleotide encoding a
polypeptide are known in the art and include isolation from genomic DNA,
preparation from cDNA, or a combination thereof. The cloning of the
polynucleotides of the present invention from such genomic DNA can be
effected, e.g., by using the well known polymerase chain reaction (PCR)
or antibody screening of expression libraries to detect cloned DNA
fragments with shared structural features. See, e.g., Innis et al., 1990,
PCR: A Guide to Methods and Application, Academic Press, New York. Other
nucleic acid amplification procedures such as ligase chain reaction
(LCR), ligated activated transcription (LAT) and nucleotide
sequence-based amplification (NASBA) may be used. The polynucleotides may
be cloned from a strain of Thielavia, or another or related organism and
thus, for example, may be an allelic or species variant of the
polypeptide encoding region of the nucleotide sequence.

[0102]The present invention also relates to isolated polynucleotides
comprising or consisting of nucleotide sequences that have a degree of
identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ
ID NO: 3 of preferably at least 60%, more preferably at least 65%, more
preferably at least 70%, more preferably at least 75%, more preferably at
least 80%, more preferably at least 85%, even more preferably at least
90%, most preferably at least 95%, and even most preferably at least 96%,
at least 97%, at least 98%, or at least 99% identity, which encode an
active polypeptide.

[0103]Modification of a nucleotide sequence encoding a polypeptide of the
present invention may be necessary for the synthesis of polypeptides
substantially similar to the polypeptide. The term "substantially
similar" to the polypeptide refers to non-naturally occurring forms of
the polypeptide. These polypeptides may differ in some engineered way
from the polypeptide isolated from its native source, e.g., artificial
variants that differ in specific activity, thermostability, pH optimum,
or the like. The variant sequence may be constructed on the basis of the
nucleotide sequence presented as the mature polypeptide coding sequence
of SEQ ID NO: 1 or SEQ ID NO: 3, e.g., a subsequence thereof, and/or by
introduction of nucleotide substitutions that do not give rise to another
amino acid sequence of the polypeptide encoded by the nucleotide
sequence, but which correspond to the codon usage of the host organism
intended for production of the enzyme, or by introduction of nucleotide
substitutions that may give rise to a different amino acid sequence. For
a general description of nucleotide substitution, see, e.g., Ford et al.,
1991, Protein Expression and Purification 2: 95-107.

[0104]It will be apparent to those skilled in the art that such
substitutions can be made outside the regions critical to the function of
the molecule and still result in an active polypeptide. Amino acid
residues essential to the activity of the polypeptide encoded by an
isolated polynucleotide of the invention, and therefore preferably not
subject to substitution, may be identified according to procedures known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (see, e.g., Cunningham and Wells, 1989, supra). In the latter
technique, mutations are introduced at every positively charged residue
in the molecule, and the resultant mutant molecules are tested for
xylanase activity to identify amino acid residues that are critical to
the activity of the molecule. Sites of substrate-enzyme interaction can
also be determined by analysis of the three-dimensional structure as
determined by such techniques as nuclear magnetic resonance analysis,
crystallography or photoaffinity labeling (see, e.g., de Vos et al.,
1992, supra; Smith et al., 1992, supra; Wlodaver et al., 1992, supra).

[0106]The present invention also relates to isolated polynucleotides
obtained by (a) hybridizing a population of DNA under very low, low,
medium, medium-high, high, or very high stringency conditions with (i)
the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3,
(ii) the genomic DNA sequence comprising the mature polypeptide coding
sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a full-length
complementary strand of (i) or (ii); and (b) isolating the hybridizing
polynucleotide, which encodes a polypeptide having xylanase activity. In
a preferred aspect, the complementary strand is the full-length
complementary strand of the mature polypeptide coding sequence of SEQ ID
NO: 1 or SEQ ID NO: 3.

Nucleic Acid Constructs

[0107]The present invention also relates to nucleic acid constructs
comprising an isolated polynucleotide of the present invention operably
linked to one or more (several) control sequences that direct the
expression of the coding sequence in a suitable host cell under
conditions compatible with the control sequences.

[0108]An isolated polynucleotide encoding a polypeptide of the present
invention may be manipulated in a variety of ways to provide for
expression of the polypeptide. Manipulation of the polynucleotide's
sequence prior to its insertion into a vector may be desirable or
necessary depending on the expression vector. The techniques for
modifying polynucleotide sequences utilizing recombinant DNA methods are
well known in the art.

[0109]The control sequence may be an appropriate promoter sequence, a
nucleotide sequence that is recognized by a host cell for expression of a
polynucleotide encoding a polypeptide of the present invention. The
promoter sequence contains transcriptional control sequences that mediate
the expression of the polypeptide. The promoter may be any nucleotide
sequence that shows transcriptional activity in the host cell of choice
including mutant, truncated, and hybrid promoters, and may be obtained
from genes encoding extracellular or intracellular polypeptides either
homologous or heterologous to the host cell.

[0113]The control sequence may also be a suitable transcription terminator
sequence, a sequence recognized by a host cell to terminate
transcription. The terminator sequence is operably linked to the 3'
terminus of the nucleotide sequence encoding the polypeptide. Any
terminator that is functional in the host cell of choice may be used in
the present invention.

[0116]The control sequence may also be a suitable leader sequence, a
nontranslated region of an mRNA that is important for translation by the
host cell. The leader sequence is operably linked to the 5' terminus of
the nucleotide sequence encoding the polypeptide. Any leader sequence
that is functional in the host cell of choice may be used in the present
invention.

[0119]The control sequence may also be a polyadenylation sequence, a
sequence operably linked to the 3' terminus of the nucleotide sequence
and, when transcribed, is recognized by the host cell as a signal to add
polyadenosine residues to transcribed mRNA. Any polyadenylation sequence
that is functional in the host cell of choice may be used in the present
invention.

[0122]The control sequence may also be a signal peptide coding sequence
that codes for an amino acid sequence linked to the amino terminus of a
polypeptide and directs the encoded polypeptide into the cell's secretory
pathway. The 5' end of the coding sequence of the nucleotide sequence may
inherently contain a signal peptide coding sequence naturally linked in
translation reading frame with the segment of the coding sequence that
encodes the secreted polypeptide. Alternatively, the 5' end of the coding
sequence may contain a signal peptide coding sequence that is foreign to
the coding sequence. The foreign signal peptide coding sequence may be
required where the coding sequence does not naturally contain a signal
peptide coding sequence. Alternatively, the foreign signal peptide coding
sequence may simply replace the natural signal peptide coding sequence in
order to enhance secretion of the polypeptide. However, any signal
peptide coding sequence that directs the expressed polypeptide into the
secretory pathway of a host cell of choice, i.e., secreted into a culture
medium, may be used in the present invention.

[0126]In a preferred aspect, the signal peptide comprises or consists of
amino acids 1 to 19 of SEQ ID NO: 2. In another preferred aspect, the
signal peptide coding sequence comprises or consists of nucleotides 1 to
57 of SEQ ID NO: 1. In another preferred aspect, the signal peptide
comprises or consists of amino acids 1 to 18 of SEQ ID NO: 4. In another
preferred aspect, the signal peptide coding sequence comprises or
consists of nucleotides 1 to 54 of SEQ ID NO: 3.

[0127]The control sequence may also be a propeptide coding sequence that
codes for an amino acid sequence positioned at the amino terminus of a
polypeptide. The resultant polypeptide is known as a proenzyme or
propolypeptide (or a zymogen in some cases). A propeptide is generally
inactive and can be converted to a mature active polypeptide by catalytic
or autocatalytic cleavage of the propeptide from the propolypeptide. The
propeptide coding sequence may be obtained from the genes for Bacillus
subtilis alkaline protease (aprE), Bacillus subtilis neutral protease
(nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic
proteinase, and Myceliophthora thermophila laccase (WO 95/33836).

[0128]Where both signal peptide and propeptide sequences are present at
the amino terminus of a polypeptide, the propeptide sequence is
positioned next to the amino terminus of a polypeptide and the signal
peptide sequence is positioned next to the amino terminus of the
propeptide sequence.

[0129]It may also be desirable to add regulatory sequences that allow the
regulation of the expression of the polypeptide relative to the growth of
the host cell. Examples of regulatory systems are those that cause the
expression of the gene to be turned on or off in response to a chemical
or physical stimulus, including the presence of a regulatory compound.
Regulatory systems in prokaryotic systems include the lac, tac, and trp
operator systems. In yeast, the ADH2 system or GAL1 system may be used.
In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger
glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may
be used as regulatory sequences. Other examples of regulatory sequences
are those that allow for gene amplification. In eukaryotic systems, these
regulatory sequences include the dihydrofolate reductase gene that is
amplified in the presence of methotrexate, and the metallothionein genes
that are amplified with heavy metals. In these cases, the nucleotide
sequence encoding the polypeptide would be operably linked with the
regulatory sequence.

Expression Vectors

[0130]The present invention also relates to recombinant expression vectors
comprising a polynucleotide of the present invention, a promoter, and
transcriptional and translational stop signals. The various nucleic acids
and control sequences described herein may be joined together to produce
a recombinant expression vector that may include one or more (several)
convenient restriction sites to allow for insertion or substitution of
the nucleotide sequence encoding the polypeptide at such sites.
Alternatively, a polynucleotide sequence of the present invention may be
expressed by inserting the nucleotide sequence or a nucleic acid
construct comprising the sequence into an appropriate vector for
expression. In creating the expression vector, the coding sequence is
located in the vector so that the coding sequence is operably linked with
the appropriate control sequences for expression.

[0131]The recombinant expression vector may be any vector (e.g., a plasmid
or virus) that can be conveniently subjected to recombinant DNA
procedures and can bring about expression of the nucleotide sequence. The
choice of the vector will typically depend on the compatibility of the
vector with the host cell into which the vector is to be introduced. The
vectors may be linear or closed circular plasmids.

[0132]The vector may be an autonomously replicating vector, i.e., a vector
that exists as an extrachromosomal entity, the replication of which is
independent of chromosomal replication, e.g., a plasmid, an
extrachromosomal element, a minichromosome, or an artificial chromosome.
The vector may contain any means for assuring self-replication.
Alternatively, the vector may be one that, when introduced into the host
cell, is integrated into the genome and replicated together with the
chromosome(s) into which it has been integrated. Furthermore, a single
vector or plasmid or two or more vectors or plasmids that together
contain the total DNA to be introduced into the genome of the host cell,
or a transposon, may be used.

[0133]The vectors of the present invention preferably contain one or more
(several) selectable markers that permit easy selection of transformed,
transfected, transduced, or the like cells. A selectable marker is a gene
the product of which provides for biocide or viral resistance, resistance
to heavy metals, prototrophy to auxotrophs, and the like.

[0134]Examples of bacterial selectable markers are the dal genes from
Bacillus subtilis or Bacillus licheniformis, or markers that confer
antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or
tetracycline resistance. Suitable markers for yeast host cells are ADE2,
HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a
filamentous fungal host cell include, but are not limited to, amdS
(acetamidase), argB (ornithine carbamoyltransferase), bar
(phosphinothricin acetyltransferase), hph (hygromycin
phosphotransferase), niaD (nitrate reductase), pyrG
(orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase),
and trpC (anthranilate synthase), as well as equivalents thereof.
Preferred for use in an Aspergillus cell are the amdS and pyrG genes of
Aspergillus nidulans or Aspergillus oryzae and the bar gene of
Streptomyces hygroscopicus.

[0135]The vectors of the present invention preferably contain an
element(s) that permits integration of the vector into the host cell's
genome or autonomous replication of the vector in the cell independent of
the genome.

[0136]For integration into the host cell genome, the vector may rely on
the polynucleotide's sequence encoding the polypeptide or any other
element of the vector for integration into the genome by homologous or
nonhomologous recombination. Alternatively, the vector may contain
additional nucleotide sequences for directing integration by homologous
recombination into the genome of the host cell at a precise location(s)
in the chromosome(s). To increase the likelihood of integration at a
precise location, the integrational elements should preferably contain a
sufficient number of nucleic acids, such as 100 to 10,000 base pairs,
preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000
base pairs, which have a high degree of identity to the corresponding
target sequence to enhance the probability of homologous recombination.
The integrational elements may be any sequence that is homologous with
the target sequence in the genome of the host cell. Furthermore, the
integrational elements may be non-encoding or encoding nucleotide
sequences. On the other hand, the vector may be integrated into the
genome of the host cell by non-homologous recombination.

[0137]For autonomous replication, the vector may further comprise an
origin of replication enabling the vector to replicate autonomously in
the host cell in question. The origin of replication may be any plasmid
replicator mediating autonomous replication that functions in a cell. The
term "origin of replication" or "plasmid replicator" is defined herein as
a nucleotide sequence that enables a plasmid or vector to replicate in
vivo.

[0138]Examples of bacterial origins of replication are the origins of
replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting
replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1
permitting replication in Bacillus.

[0139]Examples of origins of replication for use in a yeast host cell are
the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1
and CEN3, and the combination of ARS4 and CEN6.

[0140]Examples of origins of replication useful in a filamentous fungal
cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,
1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of
the AMA1 gene and construction of plasmids or vectors comprising the gene
can be accomplished according to the methods disclosed in WO 00/24883.

[0141]More than one copy of a polynucleotide of the present invention may
be inserted into a host cell to increase production of the gene product.
An increase in the copy number of the polynucleotide can be obtained by
integrating at least one additional copy of the sequence into the host
cell genome or by including an amplifiable selectable marker gene with
the polynucleotide where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
polynucleotide, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.

[0142]The procedures used to ligate the elements described above to
construct the recombinant expression vectors of the present invention are
well known to one skilled in the art (see, e.g., Sambrook et al., 1989,
supra).

Host Cells

[0143]The present invention also relates to recombinant host cells,
comprising an isolated polynucleotide of the present invention, which are
advantageously used in the recombinant production of the polypeptides. A
vector comprising a polynucleotide of the present invention is introduced
into a host cell so that the vector is maintained as a chromosomal
integrant or as a self-replicating extra-chromosomal vector as described
earlier. The term "host cell" encompasses any progeny of a parent cell
that is not identical to the parent cell due to mutations that occur
during replication. The choice of a host cell will to a large extent
depend upon the gene encoding the polypeptide and its source.

[0144]The host cell may be any cell useful in the recombinant production
of a polypeptide of the present invention, e.g., a prokaryote or a
eukaryote.

[0147]In a preferred aspect, the bacterial host cell is a Bacillus
amyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillus
stearothermophilus or Bacillus subtilis cell. In a more preferred aspect,
the bacterial host cell is a Bacillus amyloliquefaciens cell. In another
more preferred aspect, the bacterial host cell is a Bacillus clausii
cell. In another more preferred aspect, the bacterial host cell is a
Bacillus licheniformis cell. In another more preferred aspect, the
bacterial host cell is a Bacillus subtilis cell.

[0148]The bacterial host cell may also be any Streptococcus cell.
Streptococcus cells useful in the practice of the present invention
include, but are not limited to, Streptococcus equisimilis, Streptococcus
pyogenes, Streptococcus uberis, and Streptococcus equi subsp.
Zooepidemicus cells.

[0149]In a preferred aspect, the bacterial host cell is a Streptococcus
equisimilis cell. In another preferred aspect, the bacterial host cell is
a Streptococcus pyogenes cell. In another preferred aspect, the bacterial
host cell is a Streptococcus uberis cell. In another preferred aspect,
the bacterial host cell is a Streptococcus equi subsp. Zooepidemicus
cell.

[0150]The bacterial host cell may also be any Streptomyces cell.
Streptomyces cells useful in the practice of the present invention
include, but are not limited to, Streptomyces achromogenes, Streptomyces
avermitilis, Streptomyces coelicolor, Streptomyces griseus, and
Streptomyces lividans cells.

[0151]In a preferred aspect, the bacterial host cell is a Streptomyces
achromogenes cell. In another preferred aspect, the bacterial host cell
is a Streptomyces avermitilis cell. In another preferred aspect, the
bacterial host cell is a Streptomyces coelicolor cell. In another
preferred aspect, the bacterial host cell is a Streptomyces griseus cell.
In another preferred aspect, the bacterial host cell is a Streptomyces
lividans cell.

[0153]The host cell may also be a eukaryote, such as a mammalian, insect,
plant, or fungal cell.

[0154]In a preferred aspect, the host cell is a fungal cell. "Fungi" as
used herein includes the phyla Ascomycota, Basidiomycota,
Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,
Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB
International, University Press, Cambridge, UK) as well as the Oomycota
(as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic
fungi (Hawksworth et al., 1995, supra).

[0155]In a more preferred aspect, the fungal host cell is a yeast cell.
"Yeast" as used herein includes ascosporogenous yeast (Endomycetales),
basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti
(Blastomycetes). Since the classification of yeast may change in the
future, for the purposes of this invention, yeast shall be defined as
described in Biology and Activities of Yeast (Skinner, F. A., Passmore,
S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series
No. 9, 1980).

[0156]In an even more preferred aspect, the yeast host cell is a Candida,
Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
Yarrowia cell.

[0157]In a most preferred aspect, the yeast host cell is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,
Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis,
or Saccharomyces oviformis cell. In another most preferred aspect, the
yeast host cell is a Kluyveromyces lactis cell. In another most preferred
aspect, the yeast host cell is a Yarrowia lipolytica cell.

[0158]In another more preferred aspect, the fungal host cell is a
filamentous fungal cell. "Filamentous fungi" include all filamentous
forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth
et al., 1995, supra). The filamentous fungi are generally characterized
by a mycelial wall composed of chitin, cellulose, glucan, chitosan,
mannan, and other complex polysaccharides. Vegetative growth is by hyphal
elongation and carbon catabolism is obligately aerobic. In contrast,
vegetative growth by yeasts such as Saccharomyces cerevisiae is by
budding of a unicellular thallus and carbon catabolism may be
fermentative.

[0161]Fungal cells may be transformed by a process involving protoplast
formation, transformation of the protoplasts, and regeneration of the
cell wall in a manner known per se. Suitable procedures for
transformation of Aspergillus and Trichoderma host cells are described in
EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy
of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium
species are described by Malardier et al., 1989, Gene 78: 147-156, and WO
96/00787. Yeast may be transformed using the procedures described by
Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide
to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume
194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983,
Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of
the National Academy of Sciences USA 75: 1920.

Methods of Production

[0162]The present invention also relates to methods of producing a
polypeptide having xylanase activity of the present invention,
comprising: (a) cultivating a cell, which in its wild-type form produces
the polypeptide, under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide. In a preferred aspect,
the cell is of the genus Thielavia. In a more preferred aspect, the cell
is Thielavia terrestris. In a most preferred aspect, the cell is
Thielavia terrestris NRRL 8126.

[0163]The present invention also relates to methods of producing a
polypeptide of the present invention, comprising: (a) cultivating a
recombinant host cell, as described herein, under conditions conducive
for production of the polypeptide; and (b) recovering the polypeptide.

[0164]The present invention also relates to methods of producing a
polypeptide of the present invention, comprising: (a) cultivating a
recombinant host cell under conditions conducive for production of the
polypeptide, wherein the host cell comprises a mutant nucleotide sequence
having at least one mutation in the mature polypeptide coding sequence of
SEQ ID NO: 1 or SEQ ID NO: 3, wherein the mutant nucleotide sequence
encodes a polypeptide that comprises or consists of the mature
polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, respectively, and (b)
recovering the polypeptide.

[0165]In the production methods of the present invention, the cells are
cultivated in a nutrient medium suitable for production of the
polypeptide using methods well known in the art. For example, the cell
may be cultivated by shake flask cultivation, and small-scale or
large-scale fermentation (including continuous, batch, fed-batch, or
solid state fermentations) in laboratory or industrial fermentors
performed in a suitable medium and under conditions allowing the
polypeptide to be expressed and/or isolated. The cultivation takes place
in a suitable nutrient medium comprising carbon and nitrogen sources and
inorganic salts, using procedures known in the art. Suitable media are
available from commercial suppliers or may be prepared according to
published compositions (e.g., in catalogues of the American Type Culture
Collection). If the polypeptide is secreted into the nutrient medium, the
polypeptide can be recovered directly from the medium. If the polypeptide
is not secreted into the medium, it can be recovered from cell lysates.

[0166]The polypeptides may be detected using methods known in the art that
are specific for the polypeptides. These detection methods may include
use of specific antibodies, formation of an enzyme product, or
disappearance of an enzyme substrate. For example, an enzyme assay may be
used to determine the activity of the polypeptide as described herein.

[0167]The resulting polypeptide may be recovered using methods known in
the art. For example, the polypeptide may be recovered from the nutrient
medium by conventional procedures including, but not limited to,
centrifugation, filtration, extraction, spray-drying, evaporation, or
precipitation.

[0169]The present invention also relates to plants, e.g., a transgenic
plant, plant part, or plant cell, comprising an isolated polynucleotide
encoding a polypeptide having xylanase activity of the present invention
so as to express and produce the polypeptide in recoverable quantities.
The polypeptide may be recovered from the plant or plant part.
Alternatively, the plant or plant part containing the recombinant
polypeptide may be used as such for improving the quality of a food or
feed, e.g., improving nutritional value, palatability, and Theological
properties, or to destroy an antinutritive factor.

[0172]Examples of plant parts are stem, callus, leaves, root, fruits,
seeds, and tubers as well as the individual tissues comprising these
parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues,
meristems. Specific plant cell compartments, such as chloroplasts,
apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also
considered to be a plant part. Furthermore, any plant cell, whatever the
tissue origin, is considered to be a plant part. Likewise, plant parts
such as specific tissues and cells isolated to facilitate the utilisation
of the invention are also considered plant parts, e.g., embryos,
endosperms, aleurone and seeds coats.

[0173]Also included within the scope of the present invention are the
progeny of such plants, plant parts, and plant cells.

[0174]The transgenic plant or plant cell expressing a polypeptide of the
present invention may be constructed in accordance with methods known in
the art. In short, the plant or plant cell is constructed by
incorporating one or more (several) expression constructs encoding a
polypeptide of the present invention into the plant host genome or
chloroplast genome and propagating the resulting modified plant or plant
cell into a transgenic plant or plant cell.

[0175]The expression construct is conveniently a nucleic acid construct
that comprises a polynucleotide encoding a polypeptide of the present
invention operably linked with appropriate regulatory sequences required
for expression of the nucleotide sequence in the plant or plant part of
choice. Furthermore, the expression construct may comprise a selectable
marker useful for identifying host cells into which the expression
construct has been integrated and DNA sequences necessary for
introduction of the construct into the plant in question (the latter
depends on the DNA introduction method to be used).

[0176]The choice of regulatory sequences, such as promoter and terminator
sequences and optionally signal or transit sequences, is determined, for
example, on the basis of when, where, and how the polypeptide is desired
to be expressed. For instance, the expression of the gene encoding a
polypeptide of the present invention may be constitutive or inducible, or
may be developmental, stage or tissue specific, and the gene product may
be targeted to a specific tissue or plant part such as seeds or leaves.
Regulatory sequences are, for example, described by Tague et al., 1988,
Plant Physiology 86: 506.

[0178]A promoter enhancer element may also be used to achieve higher
expression of a polypeptide of the present invention in the plant. For
instance, the promoter enhancer element may be an intron that is placed
between the promoter and the nucleotide sequence encoding a polypeptide
of the present invention. For instance, Xu et al., 1993, supra, disclose
the use of the first intron of the rice actin 1 gene to enhance
expression.

[0179]The selectable marker gene and any other parts of the expression
construct may be chosen from those available in the art.

[0181]Presently, Agrobacterium tumefaciens-mediated gene transfer is the
method of choice for generating transgenic dicots (for a review, see
Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) and
can also be used for transforming monocots, although other transformation
methods are often used for these plants. Presently, the method of choice
for generating transgenic monocots is particle bombardment (microscopic
gold or tungsten particles coated with the transforming DNA) of embryonic
calli or developing embryos (Christou, 1992, Plant Journal 2: 275-281;
Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al.,
1992, Bio/Technology 10: 667-674). An alternative method for
transformation of monocots is based on protoplast transformation as
described by Omirulleh et al., 1993, Plant Molecular Biology 21: 415-428.

[0182]Following transformation, the transformants having incorporated the
expression construct are selected and regenerated into whole plants
according to methods well-known in the art. Often the transformation
procedure is designed for the selective elimination of selection genes
either during regeneration or in the following generations by using, for
example, co-transformation with two separate T-DNA constructs or site
specific excision of the selection gene by a specific recombinase.

[0183]The present invention also relates to methods of producing a
polypeptide of the present invention comprising: (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide encoding the
polypeptide having xylanase activity of the present invention under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide.

Removal or Reduction of Xylanase Activity

[0184]The present invention also relates to methods of producing a mutant
of a parent cell, which comprises disrupting or deleting a polynucleotide
sequence, or a portion thereof, encoding a polypeptide of the present
invention, which results in the mutant cell producing less of the
polypeptide than the parent cell when cultivated under the same
conditions.

[0185]The mutant cell may be constructed by reducing or eliminating
expression of a nucleotide sequence encoding a polypeptide of the present
invention using methods well known in the art, for example, insertions,
disruptions, replacements, or deletions. In a preferred aspect, the
nucleotide sequence is inactivated. The nucleotide sequence to be
modified or inactivated may be, for example, the coding region or a part
thereof essential for activity, or a regulatory element required for the
expression of the coding region. An example of such a regulatory or
control sequence may be a promoter sequence or a functional part thereof,
i.e., a part that is sufficient for affecting expression of the
nucleotide sequence. Other control sequences for possible modification
include, but are not limited to, a leader, polyadenylation sequence,
propeptide sequence, signal peptide sequence, transcription terminator,
and transcriptional activator.

[0186]Modification or inactivation of the nucleotide sequence may be
performed by subjecting the parent cell to mutagenesis and selecting for
mutant cells in which expression of the nucleotide sequence has been
reduced or eliminated. The mutagenesis, which may be specific or random,
may be performed, for example, by use of a suitable physical or chemical
mutagenizing agent, by use of a suitable oligonucleotide, or by
subjecting the DNA sequence to PCR generated mutagenesis. Furthermore,
the mutagenesis may be performed by use of any combination of these
mutagenizing agents.

[0188]When such agents are used, the mutagenesis is typically performed by
incubating the parent cell to be mutagenized in the presence of the
mutagenizing agent of choice under suitable conditions, and screening
and/or selecting for mutant cells exhibiting reduced or no expression of
the gene.

[0189]Modification or inactivation of the nucleotide sequence may be
accomplished by introduction, substitution, or removal of one or more
(several) nucleotides in the gene or a regulatory element required for
the transcription or translation thereof. For example, nucleotides may be
inserted or removed so as to result in the introduction of a stop codon,
the removal of the start codon, or a change in the open reading frame.
Such modification or inactivation may be accomplished by site-directed
mutagenesis or PCR generated mutagenesis in accordance with methods known
in the art. Although, in principle, the modification may be performed in
vivo, i.e., directly on the cell expressing the nucleotide sequence to be
modified, it is preferred that the modification be performed in vitro as
exemplified below.

[0190]An example of a convenient way to eliminate or reduce expression of
a nucleotide sequence by a cell is based on techniques of gene
replacement, gene deletion, or gene disruption. For example, in the gene
disruption method, a nucleic acid sequence corresponding to the
endogenous nucleotide sequence is mutagenized in vitro to produce a
defective nucleic acid sequence that is then transformed into the parent
cell to produce a defective gene. By homologous recombination, the
defective nucleic acid sequence replaces the endogenous nucleotide
sequence. It may be desirable that the defective nucleotide sequence also
encodes a marker that may be used for selection of transformants in which
the nucleotide sequence has been modified or destroyed. In a particularly
preferred aspect, the nucleotide sequence is disrupted with a selectable
marker such as those described herein.

[0191]Alternatively, modification or inactivation of the nucleotide
sequence may be performed by established anti-sense or RNAi techniques
using a sequence complementary to the nucleotide sequence. More
specifically, expression of the nucleotide sequence by a cell may be
reduced or eliminated by introducing a sequence complementary to the
nucleotide sequence of the gene that may be transcribed in the cell and
is capable of hybridizing to the mRNA produced in the cell. Under
conditions allowing the complementary anti-sense nucleotide sequence to
hybridize to the mRNA, the amount of protein translated is thus reduced
or eliminated.

[0192]The present invention further relates to a mutant cell of a parent
cell that comprises a disruption or deletion of a nucleotide sequence
encoding the polypeptide or a control sequence thereof, which results in
the mutant cell producing less of the polypeptide or no polypeptide
compared to the parent cell.

[0193]The polypeptide-deficient mutant cells so created are particularly
useful as host cells for the expression of native and/or heterologous
polypeptides. Therefore, the present invention further relates to methods
of producing a native or heterologous polypeptide comprising: (a)
cultivating the mutant cell under conditions conducive for production of
the polypeptide; and (b) recovering the polypeptide. The term
"heterologous polypeptides" is defined herein as polypeptides that are
not native to the host cell, a native protein in which modifications have
been made to alter the native sequence, or a native protein whose
expression is quantitatively altered as a result of a manipulation of the
host cell by recombinant DNA techniques.

[0194]In a further aspect, the present invention relates to a method of
producing a protein product essentially free of xylanase activity by
fermentation of a cell that produces both a polypeptide of the present
invention as well as the protein product of interest by adding an
effective amount of an agent capable of inhibiting xylanase activity to
the fermentation broth before, during, or after the fermentation has been
completed, recovering the product of interest from the fermentation
broth, and optionally subjecting the recovered product to further
purification.

[0195]In a further aspect, the present invention relates to a method of
producing a protein product essentially free of xylanase activity by
cultivating the cell under conditions permitting the expression of the
product, subjecting the resultant culture broth to a combined pH and
temperature treatment so as to reduce the xylanase activity
substantially, and recovering the product from the culture broth.
Alternatively, the combined pH and temperature treatment may be performed
on an enzyme preparation recovered from the culture broth. The combined
pH and temperature treatment may optionally be used in combination with a
treatment with an xylanase inhibitor.

[0196]In accordance with this aspect of the invention, it is possible to
remove at least 60%, preferably at least 75%, more preferably at least
85%, still more preferably at least 95%, and most preferably at least 99%
of the xylanase activity. Complete removal of xylanase activity may be
obtained by use of this method.

[0197]The combined pH and temperature treatment is preferably carried out
at a pH in the range of 2-4 or 9-11 and a temperature in the range of at
least 60-70° C. for a sufficient period of time to attain the
desired effect, where typically, 30 to 60 minutes is sufficient.

[0198]The methods used for cultivation and purification of the product of
interest may be performed by methods known in the art.

[0200]It will be understood that the term "eukaryotic polypeptides"
includes not only native polypeptides, but also those polypeptides, e.g.,
enzymes, which have been modified by amino acid substitutions, deletions
or additions, or other such modifications to enhance activity,
thermostability, pH tolerance and the like.

[0201]In a further aspect, the present invention relates to a protein
product essentially free from xylanase activity that is produced by a
method of the present invention.

Methods of Inhibiting Expression of a Polypeptide Having Xylanase Activity

[0202]The present invention also relates to methods of inhibiting the
expression of a polypeptide having xylanase activity in a cell,
comprising administering to the cell or expressing in the cell a
double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a
subsequence of a polynucleotide of the present invention. In a preferred
aspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or
more duplex nucleotides in length.

[0203]The dsRNA is preferably a small interfering RNA (siRNA) or a micro
RNA (miRNA). In a preferred aspect, the dsRNA is small interfering RNA
(siRNAs) for inhibiting transcription. In another preferred aspect, the
dsRNA is micro RNA (miRNAs) for inhibiting translation.

[0204]The present invention also relates to such double-stranded RNA
(dsRNA) molecules, comprising a portion of the mature polypeptide coding
sequence of SEQ ID NO: 1 or SEQ ID NO: 3 for inhibiting expression of a
polypeptide in a cell. While the present invention is not limited by any
particular mechanism of action, the dsRNA can enter a cell and cause the
degradation of a single-stranded RNA (ssRNA) of similar or identical
sequences, including endogenous mRNAs. When a cell is exposed to dsRNA,
mRNA from the homologous gene is selectively degraded by a process called
RNA interference (RNAi).

[0205]The dsRNAs of the present invention can be used in gene-silencing
therapeutics. In one aspect, the invention provides methods to
selectively degrade RNA using the dsRNA is of the present invention. The
process may be practiced in vitro, ex vivo or in vivo. In one aspect, the
dsRNA molecules can be used to generate a loss-of-function mutation in a
cell, an organ or an animal. Methods for making and using dsRNA molecules
to selectively degrade RNA are well known in the art, see, for example,
U.S. Pat. No. 6,506,559; U.S. Pat. No. 6,511,824; U.S. Pat. No.
6,515,109; and U.S. Pat. No. 6,489,127.

Compositions

[0206]The present invention also relates to compositions comprising a
polypeptide of the present invention. Preferably, the compositions are
enriched in such a polypeptide. The term "enriched" indicates that the
xylanase activity of the composition has been increased, e.g., with an
enrichment factor of at least 1.1.

[0208]The polypeptide compositions may be prepared in accordance with
methods known in the art and may be in the form of a liquid or a dry
composition. For instance, the polypeptide composition may be in the form
of a granulate or a microgranulate. The polypeptide to be included in the
composition may be stabilized in accordance with methods known in the
art.

[0209]Examples are given below of preferred uses of the polypeptide
compositions of the invention. The dosage of the polypeptide composition
of the invention and other conditions under which the composition is used
may be determined on the basis of methods known in the art.

Uses

[0210]The present invention is also directed to methods for using the
polypeptides having xylanase activity.

[0211]The polypeptides of the present invention can be used for
degradation or modification of plant cell walls or any xylan-containing
material originating from plant cells walls. Examples of various uses are
described below (see, WO 2002/18561, for other uses). The dosage of the
polypeptides of the present invention and other conditions under which
the preparation is used may be determined on the basis of methods known
in the art.

[0212]The enzymatic degradation of xylan is facilitated by full or partial
removal of the side branches. The polypeptides of the present invention
are preferably used in conjunction with other xylan degrading enzymes
such as acetylxylan esterases, arabinofuranosidases, xylosidases,
feruloyl esterases, glucuronidases, and a combination thereof, in
processes wherein xylan is to be degraded. For example, acetyl groups can
be removed by acetylxylan esterases; arabinose groups by
alpha-arabinosidases; feruloyl groups by feruloyl esterases, and
glucuronic acid groups by alpha-glucuronidases. The oligomers released by
the xylanases, or by a combination of xylanases and side
branch-hydrolyzing enzymes, can be further degraded to free xylose by
beta-xylosidases. A polypeptide of the present invention is preferably a
component of a composition comprising one or more (several) xylan
degrading enzymes. In the various uses described below, a polypeptide of
the present invention is preferably used in combination with one or more
(several) xylan degrading enzymes.

[0213]Consequently, the present invention also relates to methods for
degrading a xylan-containing material, comprising treating the
xylan-containing material with such a polypeptide having xylanase
activity. In a preferred aspect, the xylan-containing material is further
treated with a xylan degrading enzyme. The xylan degrading enzyme can be
selected from the group consisting of a an acetyxylan esterase, a
feruloyl esterase, an arabinofuranosidase, a xylosidase, a glucuronidase,
and a combination thereof.

[0214]The plant material may be degraded in order to improve different
kinds of processing, facilitate purification or extraction of components
other than the xylans, like purification of beta-glucan or beta-glucan
oligomers from cereals, improve the feed value, decrease the water
binding capacity, improve the degradability in waste water plants,
improve the conversion of, for example, grass and corn to ensilage, etc.
The polypeptides of the present invention may be used in the enzymatic
hydrolysis of various plant cell wall-derived materials or waste
materials, e.g., from paper production, or agricultural residues such as
wheat-straw, corn cobs, corn fiber, whole corn plants, nut shells, grass,
vegetable hulls, bean hulls, spent grains, sugar beet pulp, and the like.
The polypeptides may also be used for modifying the viscosity of plant
cell wall derived material. For instance, the polypeptides may be used to
reduce the viscosity of xylan-containing material, to promote processing
of viscous xylan-containing material, such as in wheat separation.

[0215]The polypeptides of the present invention may also be used with
limited activity of other xylanolytic enzymes to degrade xylans for
production of oligosaccharides. The oligosaccharides may be used as
bulking agents, like arabinoxylan oligosaccharides released from cereal
cell wall material, or of more or less purified arabinoxylans from
cereals.

[0216]The polypeptides of the present invention may also be used in
combination with other xylanolytic enzymes to degrade xylans to xylose
and other monosaccharides (U.S. Pat. No. 5,658,765). The released xylose
may be converted to other compounds.

[0217]The polypeptides of the present invention may also be used in
lignocellulosic biomass degradation or conversion to fermentable sugars
for the production of, for example, fuel, potable ethanol, and/or
fermentation products (e.g., acids, alcohols, ketones, gases, and the
like). The polypeptides are preferably used in combination with other
xylan degrading enzymes and a cellulase composition (endoglucanase(s),
cellobiohydrolase(s), and beta-glucosidase(s)).

[0218]The polypeptides of the present invention may be used together with
other enzymes like glucanases to improve the extraction of oil from
oil-rich plant material, like corn-oil from corn-embryos.

[0219]The polypeptides of the present invention may also be used in baking
to improve the development, elasticity, and/or stability of dough and/or
the volume, crumb structure, and/or anti-staling properties of the baked
product. The polypeptides may be used for the preparation of dough or
baked products prepared from any type of flour or meal (e.g., based on
wheat, rye, barley, oat, or maize). The baked products produced with a
polypeptide of the present invention include bread, rolls, baquettes and
the like. For baking purposes a polypeptide of the present invention may
be used as the only or major enzymatic activity, or may be used in
combination with other enzymes such as a lipase, an amylase, an oxidase
(e.g., glucose oxidase, peroxidase), a laccase and/or a protease.

[0220]The polypeptides of the present invention may also be used for
modification of animal feed and may exert their effect either in vitro
(by modifying components of the feed) or in vivo. The polypeptides may be
added to animal feed compositions containing high amounts of
arabinoxylans and glucuronoxylans, e.g., feed containing cereals such as
barley, wheat, rye, oats, or maize. When added to feed the polypeptide
will improve the in vivo break-down of plant cell wall material partly
due to a reduction of intestinal viscosity (Bedford et al., 1993,
Proceedings of the 1st Symposium on Enzymes in Animal Nutrition, pp.
73-77), whereby improved utilization of the plant nutrients by the animal
is achieved. Thereby, the growth rate and/or feed conversion ratio (i.e.,
the weight of ingested feed relative to weight gain) of the animal is
improved.

[0221]The polypeptides of the present invention may also be used in the
paper and pulp industry, inter alia in bleaching processes to enhance the
brightness of bleached pulps whereby the amount of chlorine used in the
bleaching stages is reduced, and to increase the freeness of pulps in the
recycled paper process (Eriksson, 1990, Wood Science and Technology 24:
79-101; Paice et al., 1988, Biotechnol. and Bioeng. 32: 235-239, and
Pommier et al., 1989, Tappi Journal 187-191). Furthermore, the
polypeptides may be used for treatment of lignocellulosic pulp so as to
improve the bleachability thereof. The treatment of lignocellulosic pulp
may be performed, for example, as described in U.S. Pat. No. 5,658,765,
WO 93/08275, WO 91/02839, and WO 92/03608.

[0222]The polypeptides of the present invention may also be used in beer
brewing, in particular to improve the filterability of wort containing,
for example, barley and/or sorghum malt (WO 2002/24926). The polypeptides
may be used in the same manner as pentosanases conventionally used for
brewing, e.g., as described by Vietor et al., 1993, J. Inst. Brew. 99:
243-248; and EP 227159. Furthermore, the polypeptides may be used for
treatment of brewers spent grain, i.e., residuals from beer wort
production containing barley or malted barley or other cereals, so as to
improve the utilization of the residuals for, e.g., animal feed.

[0223]The polypeptides of the present invention may be used for separation
of components of plant cell materials, in particular of cereal components
such as wheat components. Of particular interest is the separation of
wheat into gluten and starch, i.e., components of considerable commercial
interest. The separation process may be performed by use of methods known
in the art, conveniently a so-called batter process (or wet milling
process) performed as a hydroclone or a decanter process. In the batter
process, the starting material is a dilute pumpable dispersion of the
plant material such as wheat to be subjected to separation. In a wheat
separation process the dispersion is made normally from wheat flour and
water.

[0224]The polypeptides of the invention may also be used in the
preparation of fruit or vegetable juice in order to increase yield.

[0225]The polypeptides of the present invention may also be used as a
component of an enzymatic scouring system for textiles.

[0226]The polypeptides of the present invention may also be used in
laundry detergent applications in combination with other enzyme
functionalities.

Signal Peptide

[0227]The present invention also relates to nucleic acid constructs
comprising a gene encoding a protein, wherein the gene is operably linked
to a nucleotide sequence encoding a signal peptide comprising or
consisting of amino acids 1 to 19 of SEQ ID NO: 2 or amino acids 1 to 18
of SEQ ID NO: 4, wherein the gene is foreign to the nucleotide sequence.

[0228]In a preferred aspect, the nucleotide sequence comprises or consists
of nucleotides 1 to 57 of SEQ ID NO: 1. In another preferred aspect, the
nucleotide sequence comprises or consists of nucleotides 1 to 54 of SEQ
ID NO: 3.

[0230]The present invention also relates to methods of producing a protein
comprising (a) cultivating such a recombinant host cell under conditions
suitable for production of the protein; and (b) recovering the protein.

[0231]The protein may be native or heterologous to a host cell. The term
"protein" is not meant herein to refer to a specific length of the
encoded product and, therefore, encompasses peptides, oligopeptides, and
proteins. The term "protein" also encompasses two or more polypeptides
combined to form the encoded product. The proteins also include hybrid
polypeptides that comprise a combination of partial or complete
polypeptide sequences obtained from at least two different proteins
wherein one or more (several) may be heterologous or native to the host
cell. Proteins further include naturally occurring allelic and engineered
variations of the above mentioned proteins and hybrid proteins.

[0242]MDU2BP medium was composed per liter of 45 g of maltose, 1 g of
MgSO4.7H2O, 1 g of NaCl, 2 g of K2HSO4, 12 g of
KH2PO4, 2 g of urea, and 500 μl of AMG trace metals
solution, the pH was adjusted to 5.0 and then filter sterilized with a
0.22 μm filtering unit.

[0246]2×YT medium was composed per liter of 16 g of tryptone, 10 g
of yeast extract, 5 g of NaCl, and 15 g of Bacto agar.

Example 1

Expressed Sequence Tags (EST) cDNA Library Construction

[0247]Thielavia terrestris NRRL 8126 was cultivated in 50 ml of NNCYPmod
medium supplemented with 1% glucose in a 250 ml flask at 45° C.
for 24 hours with shaking at 200 rpm. A two ml aliquot from the 24-hour
liquid culture was used to seed a 500 ml flask containing 100 ml of
NNCYPmod medium supplemented with 2% SIGMACELL® 20 (Sigma Chemical
Co., St. Louis, Mo., USA). The culture was incubated at 45° C. for
3 days with shaking at 200 rpm. The mycelia were harvested by filtration
through a funnel with a glass fiber prefilter (Nalgene, Rochester, N.Y.,
USA), washed twice with 10 mM Tris-HCl-1 mM EDTA pH 8 (TE), and quick
frozen in liquid nitrogen.

[0248]Total RNA was isolated using the following method. Frozen mycelia of
Thielavia terrestris NRRL 8126 were ground in an electric coffee grinder.
The ground material was mixed 1:1 v/v with 20 ml of FENAZOL® (Ambion,
Inc., Austin, Tex., USA) in a 50 ml FALCON® tube. Once the mycelia
were suspended, they were extracted with chloroform and three times with
a mixture of phenol-chloroform-isoamyl alcohol 25:24:1 v/v/v. From the
resulting aqueous phase, the RNA was precipitated by adding 1/10 volume
of 3 M sodium acetate pH 5.2 and 1.25 volumes of isopropanol. The
precipitated RNA was recovered by centrifugation at 12,000×g for 30
minutes at 4° C. The final pellet was washed with cold 70%
ethanol, air dried, and resuspended in 500 ml of diethylpyrocarbonate
treated water (DEPC-water).

[0249]The quality and quantity of the purified RNA was assessed with an
AGILENT® 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto,
Calif., USA). Polyadenylated mRNA was isolated from 360 μg of total
RNA with the aid of a POLY(A)PURIST® Magnetic Kit (Ambion, Inc.,
Austin, Tex., USA) according to the manufacturer's instructions.

[0250]To create the cDNA library, a CLONEMINER® Kit (Invitrogen Corp.,
Carlsbad, Calif., USA) was employed to construct a directional library
that does not require the use of restriction enzyme cloning, thereby
reducing the number of chimeric clones and size bias.

[0251]To insure the successful synthesis of the cDNA, two reactions were
performed in parallel with two different concentrations of mRNA (2.2 and
4.4 μg of poly (A).sup.+ mRNA). The mRNA samples were mixed with a
Biotin-attB2-Oligo(dt) primer (Invitrogen Corp., Carlsbad, Calif., USA),
1× first strand buffer (Invitrogen Corp., Carlsbad, Calif., USA), 2
μl of 0.1 M dithiothreitol (DTT), 10 mM of each dNTP, and water to a
final volume of 18 and 16 μl, respectively.

[0252]The reaction mixtures were mixed and then 2 and 4 μl of
SUPERSCRIPT® reverse transcriptase (Invitrogen Corp., Carlsbad,
Calif., USA) were added. The reaction mixtures were incubated at
45° C. for 60 minutes to synthesize the first complementary
strand. For second strand synthesis, to each first strand reaction was
added 30 μl of 5× second strand buffer (Invitrogen Corp.,
Carlsbad, Calif., USA), 3 μl of 10 mM of each dNTP, 10 units of E.
coli DNA ligase (Invitrogen Corp., Carlsbad, Calif., USA), 40 units of E.
coli DNA polymerase I (invitrogen Corp., Carlsbad, Calif., USA), and 2
units of E. coli RNase H (Invitrogen Corp., Carlsbad, Calif., USA) in a
total volume of 150 μl. The mixtures were then incubated at 16°
C. for two hours. After the two-hour incubation 2 μl of T4 DNA
polymerase (Invitrogen Corp., Carlsbad, Calif., USA) were added to each
reaction and incubated at 16° C. for 5 minutes to create a
bunt-ended cDNA. The cDNA reactions were extracted with a mixture of
phenol-chloroform-isoamyl alcohol 25:24:1 v/v/v and precipitated in the
presence of 20 μg of glycogen, 120 μl of 5 M ammonium acetate, and
660 μl of ethanol. After centrifugation at 12,000×g for 30
minutes at 4° C., the cDNA pellets were washed with cold 70%
ethanol, dried under vacuum for 2-3 minutes, and resuspended in 18 μl
of DEPC-water. To each resuspended cDNA sample was added 10 μl of
5× adapted buffer (Invitrogen, Carlsbad, Calif., USA), 10 μg of
each attB1 adapter (Invitrogen, Carlsbad, Calif., USA), 7 μl of 0.1 M
DTT, and 5 units of T4 DNA ligase (Invitrogen, Carlsbad, Calif., USA).

[0253]Ligation reactions were incubated overnight at 16° C. Excess
adapters were removed by size-exclusion chromatography in 1 ml of
SEPHACRYL® S-500 HR resin (Amersham Biosciences, Piscataway, N.J.,
USA). Column fractions were collected according to the CLONEMINER®
Kit's instructions and fractions 3 to 14 were analyzed with an
AGILENT® 2100 Bioanalyzer to determine the fraction at which the
attB1 adapters started to elute. This analysis showed that the adapters
began eluting around fraction 10 or 11. For the first library fractions
6-11 were pooled and for the second library fractions 4-11 were pooled.

[0254]Cloning of the cDNA was performed by homologous DNA recombination
according to the GATEWAY® System protocol (Invitrogen Corp.,
Carlsbad, Calif., USA) using BP CLONASE® (Invitrogen Corp., Carlsbad,
Calif., USA) as the recombinase. Each BP CLONASE® recombination
reaction contained approximately 70 ng of attB-flanked-cDNA, 250 ng of
pDONR® 222, 2 μl of 5×BP CLONASE® buffer, 2 μl of TE,
and 3 μl of BP CLONASE®. All reagents were obtained from
Invitrogen, Carlsbad, Calif., USA. Recombination reactions were incubated
at 25° C. overnight.

[0255]Heat-inactivated BP recombination reactions were then divided into 6
aliquots and electroporated into ELECTROMAX® E. coli DH10B
electrocompetent cells (Invitrogen Corp., Carlsbad, Calif., USA) using a
GENE PULSER® (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) with
the following parameters: Voltage: 2.0 kV; Resistance: 200Ω; and
Capacity: 25 μF. Electrophorated cells were resuspended in 1 ml of SOC
medium and incubated at 37° C. for 60 minutes with constant
shaking at 200 rpm. After the incubation period, the transformed cells
were pooled and mixed 1:1 with freezing medium. A 200 μl aliquot was
removed for library titration and then the rest of each library was
aliquoted into 1.8 ml cryovials (Wheaton Science Products, Millville,
N.J., USA) and stored frozen at -80° C.

[0256]Four serial dilutions of each library were prepared: 1/100, 1/1000,
1/104, and 1/105. From each dilution 100 μl were plated onto
150 mm LB plates supplemented with 50 μg of kanamycin per ml and
incubated at 37° C. overnight. The number of colonies on each
dilution plate was counted and used to calculate the total number of
transformants in each library.

[0257]The first library contained approximately 5.4 million independent
clones and the second library contained approximately 9 million
independent clones.

Example 2

Template Preparation and Nucleotide Sequencing of cDNA Clones

[0258]Aliquots from both libraries described in Example 1 were mixed and
plated onto 25×25 cm LB plates supplemented with 50 μg of
kanamycin per ml. Individual colonies were arrayed onto 96-well plates
containing 100 μl of LB supplemented with 50 μg of kanamycin per ml
with the aid of a QPix Robot (Genetix Inc., Boston, Mass., USA).
Forty-five 96-well plates were obtained for a total of 4320 individual
clones. The plates were incubated overnight at 37° C. with shaking
at 200 rpm. After incubation, 100 μl of sterile 50% glycerol was added
to each well. The transformants were replicated with the aid of a 96-pin
tool (Boekel, Feasterville, Pa., USA) into secondary, deep-dish 96-well
microculture plates (Advanced Genetic Technologies Corporation,
Gaithersburg, Md., USA) containing 1 ml of MAGNIFICENT BROTH®
(MacConnell Research, San Diego, Calif., USA) supplemented with 50 μg
of kanamycin per ml in each well. The primary microtiter plates were
stored frozen at -80° C. The secondary deep-dish plates were
incubated at 37° C. overnight with vigorous agitation at 300 rpm
on a rotary shaker. To prevent spilling and cross-contamination, and to
allow sufficient aeration, each secondary culture plate was covered with
a polypropylene pad (Advanced Genetic Technologies Corporation,
Gaithersburg, Md., USA) and a plastic microtiter dish cover. Plasmid DNA
was prepared with a Robot-Smart 384 (MWG Biotech Inc., High Point, N.C.,
USA) and a MONTAGET® Plasmid Miniprep Kit (Millipore, Billerica,
Mass., USA).

[0261]Base calling, quality value assignment, and vector trimming were
performed with the assistance of PHRED/PHRAP software (University of
Washington, Seattle, Wash., USA). Clustering analysis of the ESTs was
performed with a Transcript Assembler v. 2.6.2. (Paracel, Inc., Pasadena,
Calif., USA). Analysis of the EST clustering indicated the presence of
395 independent clusters.

[0262]Sequence homology analysis of the assembled EST sequences against
the PIR and other databases was performed with the Blastx program
(Altschul et. al., 1990, J. Mol. Biol. 215:403-410) on a 32-node Linux
cluster (Paracel, Inc., Pasadena, Calif., USA) using the BLOSUM 62 matrix
(Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) From these,
246 had hits to known genes in various protein databases and 149 had no
significant hits against these databases. Among these 246 genes, 13 had
hits against well characterized homologues of glycosyl hydrolase genes.

[0263]A cDNA clone encoding a Thielavia terrestris Family 10 xylanase
(GH10A) was initially identified by sequence homology to a xylanase from
Agaricus bisporus (GenPept accession number O60206). Another cDNA clone
encoding another Thielavia terrestris Family 10 xylanase (GH10B) was
initially identified by sequence homology to a xylanase from Humicola
grisea (GenPept accession number BAA19220).

[0264]After this initial identification, clones designated Tter10D9
(GH10A) and Tter23D1 (GH10B) were retrieved from their original frozen
stock plates and streaked onto LB plates supplemented with 50 μg of
kanamycin per ml. The plates were incubated overnight at 37° C.
and a single colony from each plate was used to inoculate 3 ml of LB
medium supplemented with 150 μg of kanamycin per ml. The liquid
cultures were incubated overnight at 37° C. and plasmid DNA was
prepared from both with a BIOROBOT® 9600 (QIAGEN INC., Inc.,
Valencia, Calif., USA). Plasmid DNAs from clones Tter10D9 and Tter23D1
were sequenced again with BIGDYE® terminator chemistry as described
above, using the M13 forward primer, the M13 reverse primer, and a Poly-T
primer shown below to sequence the 3' end of the clone.
5'-TTTTTTTTTTTTTTTTTTTTTTTVN-3' (SEQ ID NO: 6), where V=G, A, C and N=G,
A, C, T.

[0265]Analysis of the deduced amino acid sequence of clones 10D9 with the
Interproscan program (Zdobnov and Apweiler, 2001, Bioinformatics 17:
847-8) showed that the amino acid sequence contained the sequence
signature of the glycosyl hydrolase Family 10. This sequence signature
known as the Pfam: PF00331 was found 28 amino acids from the starting
amino acid methionine confirming that clone Tter10D9 encoded a Family 10
glycosyl hydrolase.

[0266]Analysis of the deduced amino acid sequence of clone 23D1 showed
that this protein also contained the signature of the glycosyl hydrolase
family 10 Pfam: PF00331. The signature sequence was found 18 amino acids
from the starting amino acid methionine confirming that clone Tter23D1
encoded a Family 10 glycosyl hydrolase.

[0271]Once the identities of clones Tter10D9 and Tter23D1 were confirmed,
a 0.5 μl aliquot of plasmid DNA from each clone designated pTter10A
(FIG. 3) and pTter10B (FIG. 4) was transferred into separate vials of E.
coli TOP10 cells (Invitrogen Corp., Carlsbad, Calif., USA), gently mixed,
and incubated on ice for 10 minutes. The cells were then heat-shocked at
42° C. for 30 seconds and incubated again on ice for 2 minutes.
The cells were resuspended in 250 μl of SOC medium and incubated at
37° C. for 60 minutes with constant shaking at 200 rpm. After the
incubation period, two 30 μl aliquots were plated onto LB plates
supplemented with 50 μg of kanamycin per ml and incubated overnight at
37° C. The next day a single colony was picked from each
transformation and streaked onto three 1.8 ml cryovials containing about
1.5 mls of LB agarose supplemented with 50 μg of kanamycin per ml. The
vials were sealed with PETRISEAL® (Diversified Biotech, Boston Mass.,
USA) and deposited with the Agricultural Research Service Patent Culture
Collection, Northern Regional Research Center, Peoria, Ill., USA, as NRRL
B-50079 and NRRL B-50080 with a deposit date of Nov. 30, 2007.

Deposits of Biological Material

[0272]The following biological materials have been deposited under the
terms of the Budapest Treaty with the Agricultural Research Service
Patent Culture Collection (NRRL), Northern Regional Research Center, 1815
University Street, Peoria, Ill., 61604, USA, and given the following
accession numbers:

[0273]The strains have been deposited under conditions that assure that
access to the culture will be available during the pendency of this
patent application to one determined by foreign patent laws to be
entitled thereto. The deposits represent a substantially pure culture of
the deposited strains. The deposits are available as required by foreign
patent laws in countries wherein counterparts of the subject application,
or its progeny are filed. However, it should be understood that the
availability of a deposit does not constitute a license to practice the
subject invention in derogation of patent rights granted by governmental
action.

[0274]The invention described and claimed herein is not to be limited in
scope by the specific aspects herein disclosed, since these aspects are
intended as illustrations of several aspects of the invention. Any
equivalent aspects are intended to be within the scope of this invention.
Indeed, various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. In the case of conflict,
the present disclosure including definitions will control.